Serotype-Specific Regulation of Dengue Virus NS5 Protein Subcellular Localization

Abstract

Dengue virus (DENV) nonstructural protein 5 (NS5), consisting of methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, is critical for viral RNA synthesis within endoplasmic reticulum-derived replication complexes in the cytoplasm. However, a significant proportion of NS5 is localized to the nucleus of infected cells for DENV2, 3, and 4, whereas DENV1 NS5 is localized diffusely in the cytoplasm. We still have an incomplete understanding of how the DENV NS5 subcellular localization is regulated. Within NS5, two putative nuclear localization signal (NLS) sequences have been identified: NLS_Central residing in the palm of the RdRp domain as well as the recently discovered NLS_C-term residing in the flexible region at the C-terminal of the RdRp domain. We have previously shown that DENV2 NS5 nuclear localization can be significantly reduced by single-point mutations to the NLS_C-term. Here, we present biochemical, virological, and structural data demonstrating that the relative importance of either NLS in NS5 nuclear localization is unique to each of the four DENV serotypes. DENV1 NS5's cytoplasmic localization appears to be due to a functionally weak interaction between its NLS_Central and importin-α (IMPα), while DENV2 NS5 is almost exclusively nuclear through its NLS_C-term's strong interaction with IMPα. Both NLSs of DENV3 NS5 appear to contribute to directing its nuclear localization. Lastly, in the case of DENV4, the regulation of its NS5 nuclear localization remains an enigma but appears to be associated with its NLS_C-term.

Keywords: dengue virus serotypes; importin; importin-α cocrystal structure; nuclear localization signal.

Figure 1

Impact of NLS_c-term P_–1 mutations on the subcellular localization of DENV1–4 NS5 in infected cells. (A) Huh7 cells were infected with WT and NLS_C-term P_–1 mutant DENV (GenBank accession numbers: DENV1 (EU081230), DENV2(EU081177), DENV3 (EU081190), and DENV4 (GQ398256)) at MOI = 1 and fixed at 24 hpi. Cells were analyzed for the presence of NS5 (green) and dsRNA (red), with nuclei stained by DAPI (blue). Digitized images were captured using a Zeiss LSM 710 upright confocal microscope with 63× oil immersion lens. The scale bar represents 50 pm. (B) Image analysis using ImageJ software was performed on digitized images of ≥20 cells to determine F_n/c for each of the viruses from (A). F_n/c values were quantified and plotted, with each point representing a quantified cell. The dashed line indicates F_n/c = 1, whereby equivalent levels of nuclear and cytoplasmic NS5 are quantified. The Kruskal–Wallis nonparametric test was used to test for statistical significance. Results are from two to three independent experiments.

Figure 2

Binding affinity of DENV NS5 NLS_central and NLS_C-term to IMPα is serotype-specific. (A) Sequence alignment of NLS_central and NLS_C-term for DENV1–4 and related flaviviruses. Residues in red have been shown to bind to the major groove of the host receptor lmportin-α in NLS peptide-lmportin α cocrystals in this (E) and previous work.⁻ The indicated cNLS scores were computed by cNLS Mapper with full-length DENV1–4 NS5 as input. The GenBank accession numbers are as follows: DENV1 (EU081230), DENV2 (EU081177), DENV3 (EU081190), DENV4 (GQ398256), DENV4 H241 (AY947539), ZIKV (KJ776791), YFV (NC 002031.1), WNV (NC_001563.2), JEV (M55506), USUV (MN813491), and TMUV (KM233707). (B) NLS_central resides in the N-terminal region of the RdRp, close to the enzymatic site, while the NLS_C-term is in the flexible C-terminal region of the RdRp. (C) Direct binding between the indicated FITC-labeled DENV NLS peptides and IMPα isoforms was assessed by fluorescence polarization assay. Curves were plotted in GraphPad Prism from three independent experiments. (D) Strong correlation between the strength of NLS_central or NLS_C-term as scored by cNLS Mapper and in vitro NLS/IMPα binding affinity. >ULOQ = above upper limit of quantitation. (E) Structure of IMPα bound to NLS_central of DENV1, DENV3, and ZIKV, resolved to 2.75, 2.1, and 2.2 Å, respectively, as well as an overlay of all three. See Table S5 for refinement statistics. The schematic highlights the hydrogen bonds and salt bridges (in boldface) forming between residues of IMPα and the corresponding NLS_central. ZIKV NLS/IMPα structure obtained from PDB: 5W41.

Figure 3

Varying NLS contributions to DENV1 and DENV3 NS5 subcellular localization in transfected cells. (A) Huh7 cells were transfected with NS5-FLAG constructs as indicated and fixed at 24 hpt (GenBank accession numbers: DENV1 (EU081230), DENV2 (EU081177), and DENV3 (EU081190)). Cells were analyzed for NS5 (green) and FLAG (red) to verify proper folding of NS5, with nuclei stained by DAPI (blue). The scale bar represents 50 μm. (B) Image analysis using ImageJ software was performed on digitized images of ≥20 cells to determine F_n/c. F_n/c values were quantified and plotted, with each point representing a quantified cell. The dashed line indicates F_n/c = 1 whereby equivalent levels of nuclear and cytoplasmic NS5 are quantified. The Kruskal–Wallis nonparametric test was used to test for statistical significance. Results are from two to three independent experiments.

Figure 4

DENV4 NS5 subcellular localization differs between infected and transfected cells. (A) Huh7 cells were transfected with FLAG-tagged pEF-Ub DENV2 and DENV4 NS5 plasmids (GenBank accession numbers: DENV2 (EU081177) and DENV4 (GQ398256)) and fixed and stained as in Figure 3A. The scale bar represents 50 μm. (B) Image analysis of transfected NS5 (A) and NS5 during infection (Figure 1A) is performed as in Figure 1. F_n/c values were quantified and plotted as violin plots to show the spread of F_n/c for each virus. Each point refers to a single cell. The dashed line indicates F_n/c = 1, whereby equivalent levels of nuclear and cytoplasmic NS5 are quantified. The median is indicated by a yellow line. The Kruskal–Wallis nonparametric test was used to test for statistical significance. Results are from two to three independent experiments.

Figure 5

Role of NLS_c-term in DENV4 NS5 nuclear localization. (A) WebLogo analysis of DENV4 sequences (375 sequences obtained from the virus variation resource database). Red triangles indicate residues predicted by MusiteDeep to be phosphorylated. The residue in pink is the P_–1 proline that is important for NLS_C-term/IMPα binding, while the blue residues are the corresponding P₁–P₅ residues identified as NLS_c-term binding determinants in DENV2 and 3 NS5. (B) DENV infectious clones were generated with the indicated mutations to investigate the role of NLS_c-term in DENV4 NS5 subcellular localization. DENV2 residues are in red. (C) Huh7 cells were infected with the DENV2 and DENV4 WT and mutant viruses as indicated (GenBank accession: DENV2 (EU081177) and DENV4 (GQ398256)). Cells are fixed with 24 hpi and stained as in Figure 1. The scale bar represents 50 μm. The panels for DENV2 P884T, DENV4 WT, and DENV4 P885T are as in Figure 1. (D) Image analysis is performed as in Figure 4B, and F_n/c values are quantified and plotted as in Figure 4B. The dashed line indicates F_n/c = 1, whereby equivalent levels of nuclear and cytoplasmic NS5 are quantified. Median intensity is indicated by a yellow line. The Kruskal–Wallis nonparametric test was used to test for statistical significance. Results are from two to three independent experiments.